Hepatitis C virus (HCV) is a RNA virus that chronically infects ~3% of the world population leading to cirrhosis and/or hepatocellular carcinoma in ~20% of those infected. Current treatments based on interferon-1 and ribavirin fail to cure the majority of people chronically infected with genotype 1 HCV, and rapidly emerging resistance to new direct-acting antivirals is likely to become a significant problem. By contrast treatment of acute infection has a far better prognosis, and at least 20% of acute HCV cases spontaneously resolve without intervention. A better understanding of which innate immune pathways lead to viral clearance during the acute phase may therefore aid the rational design of future immunomodulatory therapies. In order to establish chronicity, HCV has evolved to evade both the innate and adaptive immune systems. At the earliest steps of viral sensing, the HCV NS3-4A protease cleaves two adaptor molecules, MAVS and TRIF, rendering both RIG-I and TLR3 pathways ineffective and preventing the induction of interferons. In order to interfere downstream from the interferon response, HCV activates PKR, which leads to translational down-regulation of interferon stimulated genes (ISGs) through activation of eIF21. The relative importance and interplay of these evasion strategies is not understood. Research of HCV has been hampered by a paucity of robust in vitro culture systems of primary hepatocytes and by restriction of viral tropism to humans and chimpanzees. While murine hepatocytes cannot support HCV infection, immunodeficient mice with liver injury can be xenografted with human hepatocytes that expand over time and allow for infection with HCV. We propose to use two recently developed in vitro culture systems to investigate the relative contributions of the RIG-I and TLR3 viral sensing pathways and of PKR activation in primary human hepatocytes, and further study these pathways in a new mouse model based on FAH- deficient liver injury mice. To investigate the RIG-I and TLR3 pathways, hepatocytes will be transduced with NS3-4A cleavage-resistant forms of MAVS and TRIF. The role of PKR will be studied using knock down approaches and transduction of primary hepatocytes with eIF21 mutants. After infection, viral spread, persistence, IRF3 translocation and ISG induction will be measured. These studies will hopefully shed light on the relative contributions of these innate immune pathways to antiviral immunity in primary hepatocytes. Results obtained by these experiments can help prioritize which immunomodulatory therapies should best be pursued into clinical development.